A deoxyribonucleic acid (DNA) constitutes the genome carrying the information of life. A ribonucleic acid (RNA) is an important biological polymer which receives such information and is involved in protein biosynthesis and the like in the body. The ribonucleic acid is largely divided into a messenger RNA (mRNA), a transfer RNA (tRNA) and a ribosomal RNA (rRNA), and each has distinct properties. There are some viruses that utilize a ribonucleic acid as the genome carrying the information of life.
An analysis of ribonucleic acid provides extremely important information for the fields of biochemistry, genetic engineering, clinical diagnostics and the like. Isolation of ribonucleic acid from a biological material is an essential step for such analysis. It is necessary to use a ribonucleic acid having a highest possible purity to achieve good results in analyses, such as northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR) and the like, which are routinely used in these fields.
In generality, a ribonucleic acid cannot be extracted without rupture of cells, during which stage a ribonucleic acid is obtained in a mixture with protein, lipid, sugar, deoxyribonucleic acid and the like. Inasmuch as ribonucleic acid is easily degraded by a ribonuclease universally found in living organisms, it is isolated in the presence of a protein denaturing agent or in an organic solvent, thereby weakening the activity of the ribonuclease. The most predominantly used for this end is a so-called AGPC method (Analytical Biochemistry, 162: 156-159 (1987)), which comprises (1) extracting a biological material with a guanidine thiocyanate solution, successively adding an acidic solution, a phenol solution and a chloroform solution, (2) centrifuging the resulting mixture to separate proteins denatured with phenol and insolubilized deoxyribonucleic acids, into an intermediate layer between an organic layer and an aqueous layer, (3) adding isopropanol to the aqueous layer to insolubilize a ribonucleic acid therein, and (4) selectively precipitating the ribonucleic acid alone by centrifugation. The AGPC method is advantageous in that it can isolate ribonucleic acid relatively easily and efficiently, as compared to other methods involving ultracentrifugation for isolating ribonucleic acid. However, it requires a poisonous substance such as phenol and chloroform, as well as a rather time-consuming step such as isopropanol precipitation, which in turn gives rise to a need of a safer and time-saving method when a number of samples are to be treated at the same time in common research institutions.
In the meantime, a different, simple and convenient method for extraction of nucleic acid has been proposed by Boom et al. (J. Clin. Microbiol., 28(3):495-503 (1990)), which uses silica particles as a nucleic acid-binding carrier. This method includes (1) mixing a biological material, a neutral solution consisting of guanidine thiocyanate, EDTA and Triton X-100, and a nucleic acid-binding solid phase (silica) to bind the nucleic acid to said solid phase, (2) separating the nucleic acid-bound solid phase from a liquid phase, (3) washing said solid phase with a wash solution containing guanidine thiocyanate, (4) washing said solid phase with 70% ethanol, (5) washing the solid phase with acetone, followed by drying thereof, and (6) eluting the nucleic acid with an eluent. This method characteristically permits isolation of nucleic acid without the use of a poison such as phenol, or concentration with isopropanol. The ribonucleic acid obtained by this method contains a large amount of deoxyribonucleic acid, which renders this method unsuitable for isolation of ribonucleic acid at high purities.
As a different isolation method of nucleic acid using a carrier such as silica particles, there has been known a method comprising adsorbing a nucleic acid in an agarose gel onto the surface of glass particles in an NaI solution and separating the nucleic acid from a liquid phase (Proc. Natl. Acad. Sci. USA, 76: 615 (1979)). What is common to these methods is that silica and nucleic acid are bound in a neutral solution containing a chaotropic ion (i.e., monovalent anion having greater ionic radius), such as iodide ion and thiocyanate ion. These methods, however, mainly aim at isolation of deoxyribonucleic acid, wherein ribonucleic acid may be isolated yet only in low yields, and isolation of ribonucleic acid alone is not attainable. Again, these methods are unsuitable for isolation of ribonucleic acid.
Another isolation method (lithium precipitation method) of ribonucleic acid has been reported, which utilizes a chemical property that addition of a lithium ion to an aqueous ribonucleic acid solution leads to insolubilization of ribonucleic acid (Molecular Cloning, 2nd ed., 1.40 (1989)). This method, nevertheless, requires centrifugation at high rpm to precipitate ribonucleic acid. Thus, development of an isolation method of ribonucleic acid which is free of such difficulties has been desired.